« Previous | Contents | Next »
Listen
ANNEX 5
Contents
ANNEX 5 A. BTV-8 and the threat posed by other BTV serotypes.
ANNEX 5 B. Costs of Testing
(i). Antibody ELISA testing.
(ii) Real-time PCR testing to detect BTVRNA ('group' specific assay).
(iii) Virus Isolation of Bluetongue virus and serotyping by SNT or VNT.
(iv) Identification of Bluetongue virus serotype by conventional RT- PCR.
ANNEX 5 C. Vertical Transmission
ANNEX 5 A BTV-8 and the threat posed by other BTV serotypes.
Peter Mertens - IAH Pirbright,
The ' BTV - Scotland' project is specifically designed to assess risks and model the spread of the northern European strain of BTV-8 to Scotland. This 'spread' could occur either as a gradual movement of the virus northwards from infected regions in the south of England, Wales or Ireland, or could result from a more sudden and long distance 'jump', as has been seen on several occasions in mainland Europe (possibly as a result of infected animal and/or insect movements).
However, there are 24 distinct serotypes of the bluetongue virus ( BTV) and multiple strains exist within each 'type' (Maan et al, 2007), many of which can cause severe disease in ruminants (particularly sheep). The events of 2006 onwards show that local Culicoides populations can support the replication, transmission and spread of the bluetongue virus in northern Europe, including the UK. Therefore the whole of Europe must now be considered at 'high risk' of disease outbreaks caused by BTV, and potentially by certain other arboviruses.
The current series of European bluetongue outbreaks started in the Mediterranean region, with the introduction of BTV-9 into the Greek island of Lesbos in 1998. In subsequent years there have been multiple introductions of 8 new strains belonging to 6 of the 24 BTV 'serotypes', with new introductions every year except 2002 (see www.reoviridae.org/dsRNA_virus_proteins/ReoID/BTV-mol-epidem.htm).
BTV is transmitted by biting midges of the genus Culicoides. In the Mediterranean region and North Africa, the major vector species is thought to be Culicoides imicola. This may explain why some of the outbreaks of the disease in southern Europe have remained restricted to regions where this insect species is present. However, since the 1980s the distribution of C.imicola has increased in the Mediterranean region, coinciding with climate change. This has allowed an overlap with other more northerly (Palaearctic) Culicoides species (including members of the C.obsoletus and C. pulicaris complexes), that are abundant and widespread across northern Europe, including the UK. These changes have also provided an opportunity for the virus to colonise (and potentially adapt to) these northerly insect species as novel vectors, leading to transmission of certain BTV strains in regions where C. imicola is absent.
The most dramatic of the European outbreaks has been caused by the arrival of an African strain of BTV-8 in the Netherlands during August -July 2007, the first time this serotype had ever been recorded in Europe. It is still unclear exactly how this African virus arrived in the region, far from any known cases of BTV-8 infection. Indeed, without clear information concerning the route and mechanism of entry it is very difficult to close this particular 'door' to prevent other 'sudden' arrivals of novel bluetongue viruses anywhere in Europe, including the UK or Scotland. Indeed such introductions may have happened periodically in the past, and it is only now with climate change that these viruses can survive, replicate in the insect vectors and be transmitted, causing devastating disease outbreaks in the naïve and highly susceptible host populations in Europe.
During 2007, an African strain of BTV-1 spread across the Iberian Peninsula, from Morocco, through to southern France. This virus is now in a region of France containing the northern European vectors, suggesting that it is likely to continue its movement towards the UK during the summer of 2008. It is also possible that other viruses (like the European strain of BTV-8) may 'parachute' in from further afield.
Although this project is limited to a single bluetongue serotype ( BTV-8), it does provide a paradigm for the introduction, spread, persistence and costs of a BT outbreak that could potentially be caused by any serotype. The different BTV serotypes do not cross protect, and could in practice be considered as the causes of different diseases. However, in reality different BTV strains can interact, exchanging genome segments, and after multiple serial infections causing some cross-protection, although this would only be evident in infections of an individual animal by a third or subsequent serotype.
ANNEX 5 B Costs of testing
Based on a Paper from the CRL, IAH Pirbright describing cost of BTV testing and lab testing capacity in a 'non-outbreak' scenario, prepared by Chris Oura - 26 th November 2007; updated for molecular typing by Peter Mertens 18 th March 2008
Background: At IAH Pirbright there are 7 full-time staff working on diagnostics within the Non-Vesicular Reference labs. These laboratories include the FOA World, OIE and national reference labs for Morbilliviruses, the OIE and national reference lab for African Swine fever virus, the OIE and national reference labs for capripox viruses (sheep pox, goat pox and lumpy skin disease), the OIE, EC and national reference laboratory for bluetongue and the OIE and national reference lab for African Horse sickness. We receive many samples into these labs from around the world but also receive samples from the UK.
Molecular typing of BTV, AHSV and EHDV, also involves the Arbovirology Research Group, which includes a further four staff members involved in virus strain identification and molecular epidemiology.
Note - If bluetongue testing is to be shared between IAH Pirbright and VLA Weybridge, it may be important to reach a joint decision on testing costs. This document only explains the current testing costs at IAH Pirbright.
testing:ELISA1. Antibody
Ongoing Commercial charges for BTV antibody testing by competition ELISA (using the Pourquier assay kit): First sample - £51; Samples 2-50 - £16; Samples 51-100 - £13; Samples 101+ - £8
Note: Antibody ELISA testing on samples from imported animals since the start of the BTV-8 incursion into northern Europe in 2006, has been at the expense of IAH Pirbright. No extra money for this testing has been supplied by DEFRA. Around 5000 samples have been tested from imported animals and this testing has cost IAH Pirbright around £25,000 in reagents and staff time. The CRL has also carried out all the testing for Scotland, Northern Ireland and Wales without charge.
The cost of testing depends entirely on how many samples are tested at one time. If fewer samples are tested the individual tests become significantly more expensive, simply because as it takes a similar amount of staff time to process 20 as well as 40 samples. For maximum efficiency and the lowest cost we need to test 'full plates'. The costs (including staff time and reagent cost) are given below for antibody ELISA using a 'full plate' of samples. We presently carry out all testing in duplicate. However, to reduce the cost of the test, samples could be tested individually, although this slightly decreases the reliability of the test.
Actual costs per sample (with no profit margin) for BTV antibody ELISA detection (full plate of samples)
Staff costs per hour at FEC = ~ £75 per hour
Duplicate testing by ELISA = £6.00 per sample
Single testing by ELISA = £4.00 per sample
Testing capacity for BTV antibody ELISA testing at IAH Pirbright:
The capacity for ELISA testing depends on many factors. The limiting factor is the processing of the samples prior to testing as the ELISA test is relatively quick and easy to perform. If samples come in large batches it is much easier and quicker to process the samples than when samples are submitted in small batches. Our capacity for ELISA testing also depends how many PCR tests are being performed at the same time. Taking into consideration the staff that we have in the reference lab at the present time we could test the following amount of samples:
For testing in duplicate: 300 samples a day (1500 samples a week)
For testing singly: 400 samples a day (2,000 samples a week)
Note: If the samples are submitted in small batches capacity would reduce.
('group' specific assay).RNABTV testing to detect PCR2. Real-time
Ongoing Commercial charges for BTVRNA detection by real-time RT- PCR: Samples 1-5: £60 per sample; Samples 6-20 - £40 per sample; Samples 20+ - £20 per sample.
Note: All testing of samples from imported animals since the 2006 incursion of BTV-8 into northern Europe has been at the expense of IAH Pirbright. No extra money for this testing has been supplied by DEFRA. The primary test is based on that described by Shaw et al (2007) Around 5000 samples have been tested by RT- PCR from imported animals and this testing has cost IAH Pirbright around £60,000 in reagents and staff time. The cost of testing depends on how many samples are tested at one time. The fewer samples tested the more expensive the test. For maximum efficiency we need to test full plates. The costs (including staff time and reagent cost) are given below for RT- PCR with a full plate of samples. We presently carry out all RT- PCR testing on samples from imported animals processed singly, and all UK report case samples are processed in duplicate.
Note: It is important to note that because vaccinated animals will give a positive test result by the Pourquier ELISA, the real time RT- PCR may become the only, current major test system, that can be used to identify infected animals.
Cost per sample (with no profit margin) for BTV real-time RT- PCR (full plate of samples).
Staff costs per hour at FEC = ~ £75 per hour
Duplicate testing: = £20 per sample
Single testing = £12 per sample
Testing capacity for real-time RT- PCR testing at IAH Pirbright:
The capacity for PCR testing depends on how many ELISA tests are being performed at the same time. Taking into consideration the staff that we have in the reference lab at the present time we could test the following amount of samples:
For testing in duplicate: 80 samples a day (400 samples a week);
For testing singly: 160 samples a day (800 samples a week)
Note: If the samples are submitted in small batches capacity would reduce. Samples submitted for movement regulations are tested singly.
VNT or SNT3. Virus Isolation of Bluetongue virus and serotyping by
Virus isolation is a lengthy and relatively slow procedure and should not therefore be carried out as a routine test for the diagnosis of BTV. The real time RT- PCR test that was developed at IAH Pirbright (Shaw et al 2007) is more sensitive and more reliable, although it detects viral RNA rather than whole infectious virus.
The virus isolation testing procedure is complex and expensive, involving isolation on KC cells (a cell line derived from Culicoides varipennis, or injection of material into embryonated hen eggs, then passage the virus in tissue culture (e.g. BHK or Vero cells) prior to confirming the presence and identity of the virus by RT- PCR or ELISA. Although this method is too expensive to carry out routinely, it was previously been used for 'typing'virus isolates and still has value as a 'gold standard' for virus serotype identification.
Serological typing of BTV isolates in virus neutralisation assays ( VNT) requires access to standardised antisera for all 24 BTV types, reagents that are difficult to prepare and in short supply. These assays take ~ one week to complete and may need repetition. They also involve a great deal of staff time and are therefore costed at £1500 per virus sample tested (after virus isolation has already been completed).
Similar serum neutralisation assays ( SNT) can be used to determine the specificity of test antisera by reacting them with reference strains of the 24 BTV types. These are also time consuming (~one week), laborious, may need repetition and would also cost £1500 per serum tested. The SNT require access to pre-titrated preparation of all 24 BTV reference strains, and can give high levels of cross-reactions with sera from areas where more than one type is circulating.
If further details of methods (and costs) used for virus isolation of BTV and VNT or SNT are required, these could be obtained from the CRL but these assays are very expensive.
PCR- RT4. Identification of Bluetongue virus serotype by conventional
Serotype is controlled by outer capsid protein VP2, which is encoded by segment 2 of the Bluetongue virus genome (Maan et al 2007). It is therefore possible to identify the virus type by sequence analyses of Seg-2 (Maan et al 2007), or by conventional RT- PCR assays using primers directed against Seg-2 (Mertens et al 2007).
These are currently conventional and gel based assays and the initial RT- PCR assay uses at least two primer sets for each BTV 'type'. The costs of reagents and staff time are estimated at £1500 (including RNA extraction, RT- PCR assays, gel electrophoresis, photography and report preparation) for each sample. Initial sequence analysis of a positive band, followed by phylogenetic analysis to determine the identity of the virus strain within a specific serotype, will cost a further £1500. These costs are based on FEC but non-profit making.
Serotyping viruses by these methods is expensive and would not be carried out routinely for multiple samples. Molecular typing can be conducted on a real time RT- PCR positive blood sample, or virus isolate. It has the major advantage that it will give positive identification of each type and can be used to detect multiple serotypes in a single sample. It would currently be possible to type up to 3 samples per person per day.
ANNEX 5 C Vertical transmission
(extract from document prepared by Philip Mellor, Chris Oura, Karin Darpel and Peter Mertens - IAH Pirbright, circulated to Brussels and CVOs - March 2008)
Until recently most scientific authorities agreed that transplacental infection of the ruminant foetus by BTV, from a dam infected during pregnancy, only occurred when tissue culture passaged ( TC) virus was used. In such cases resorption, abortion, birth of weak or deformed offspring, or birth of viraemic offspring could result. Work at IAH-Pirbright (Gibbs et al 1979) using TC virus has demonstrated the birth of lambs that were viraemic for up to 60 days post-parturition. As the dams were infected at around 60-70 days of gestation, this means that there was a period of approximately 145 days between infection of the dam and the end of viraemia in the lamb. Such a time would easily cover the period from the end of one BTV-transmission season (December) to the start of the next (April) in northern Europe. However, recent observations in northern Europe (in Holland, Belgium and the UK) indicate that ruminant offspring that are weak, still born and PCR positive for BTV, are now being born to dams infected in 2007 with BTV. This is a new finding since such events are the result of field infections with a wild type virus strain, not infections with TC virus. This phenomenon requires further investigation to confirm its occurrence and frequency as, it could account for the widespread over wintering that was detected in northern Europe in 2006 to 2007.
During January 2008 (in the vector-free season) eight pregnant BTV-seropositive but RT- PCR negative animals were imported from the Netherlands into Northern Ireland (which was BTV free). Although these animals were also PCR-negative for BTV at 12 and 42 days post-importation, three of the calves born to these animals in Northern Ireland were shown to be infected with BTV by real-time RT- PCR and one calf was positive by virus isolation (virus isolation results pending on the other 2 calves) soon after it was born, demonstrating vertical transmission. The infection was also likely to have been passed to two previously sero-negative and RT- PCR negative animals that had been housed in the same building, indicating the possibility of horizontal transmission in the absence of any detectable numbers of adult vector insects.
The data from Northern Ireland not only demonstrate trans-placental transmission of BTV from dam to calf, but also provide strong evidence for horizontal (possibly mechanical or oral transmission) of the virus. Once BTV has been passed trans-placentally, it may persist in calves at significant levels, possibly indicating that the calf is immuno-compromised, or that the virus is cell-associated and protected from circulating antibodies. Cell-association is a widely recognised aspect of BTV infection. The length of time from initial infection of the dam to the end of viraemia in the calf is significant and is considered likely to be long enough to span the winter. Transmission of the virus to other animals, in the absence of adult insect vectors, may also extend the overall infection period - thus collectively providing an efficient over-wintering mechanism.
Indeed, it was in late 2007 that staff of the CRL and the Arbovirology programme at IAH Pirbright became convinced, contrary to previous scientific opinion, that there was evidence from the UK and other parts of northern Europe for overwintering of BTV in the field via trans-placental transmission through ruminant hosts. BBSRC was approached during early 2008 to request emergency funding to carry out an intensive investigation into the BTV overwintering phenomenon, the work being designed specifically to identify the mechanism or mechanisms involved. This proposal was accepted by the BBSRC in Feb 2008 and work has now commenced, providing early indications that the observations from Northern Ireland are not unusual.
ANNEX 5 References
Gibbs EP, Lawman MJ, Herniman KA. (1979) Preliminary observations on transplacental infection of bluetongue virus in sheep-a possible overwintering mechanism. Res Vet Sci. 27, 118-120.
Maan S., Maan N.S, Samuel A.R., Rao S, Attoui, H., & Mertens P.P.C (2007) Analysis and Phylogenetic Comparisons of Full-Length VP2 Genes of the Twenty-Four Bluetongue Virus Serotypes. Journal of General Virology 88:621-630.
Mertens, P.P.C., Maan N. S., Prasad, G., Samuel A.R., Shaw A., Potgieter, A.C., Anthony, S. J., and Maan S. (2007). The design of primers and use of RT- PCR assays for typing European BTV isolates: Differentiation of field and vaccine strains Journal of general Virology 88, 2811-2823.
Shaw A.E. , Monaghan, P. , Alpar, H.O. , Anthony, S. , Darpel, K.E. a, Batten, C.A. , Carpenter, S., Jones, H. , Oura, C.A.L. , King, D.P. , Elliot, H., Mellor P.S. Mertens, P.P.C. (2007) Development and validation of a real-time RT- PCR assay to detect genome bluetongue virus segment 1. Journal of Virological Methods. 145, 115-26.
« Previous | Contents | Next »